In vitro ruminal degradability of wheat straw cultivated with white-rot fungi adapted to mushroom farming conditions

Biological treatment of cereal straw for ruminant nutrition purposes might present an environmentally friendly option of valorizing a widely available by-product of grain production for farming systems with low external input. Several strains of white-rot fungi have been selected in the past under mostly controlled laboratory conditions for their capacity of lignin degradation. The study adapted to conditions on farm for upscaling purposes. The development of the in vitro straw digestibility with two different moistening pre-treatments and inoculated with three different fungi species, namely Pleurotus ostreatus, Ceriporiopsis subvermispora and Volvariella volvacea, was determined up to 42 days of fermentation with five sampling times. The effect of physical straw pre-treatments on nutritional parameters was evaluated. The neutral detergent fiber digestibility (NDFD30h), enzymatically soluble organic substance (ELOS) and the gas production (Hohenheim Feed value Test, HFT) as indicators for in vitro ruminal degradability decreased over time independent of the fungus: HFT, ELOS and NDFD30h by up to 50, 35 and 30% of the original straw. Remoistening and autoclaving the straw increased the gas production significantly by 2.6 mL/200 g dry matter (DM), and ELOS and NDFD30h by 45 and 51 g/kg DM compared to the original straw (34.9 mL/200 mg DM, 342 g/kg DM, 313 g/kg NDF).

Within 14 days of fermentation the remoistened treatments lost around 108 g/kg DM (Trial 2 and 3), which was similar to the drained straw inoculated with PO93 (Trial 1) (excluding losses by draining) (Fig. 1a,b). Losses of the drained C. subvermispora treatment were comparably low at that point (29 g/kg DM), but increased to 241 g/kg by day 28 (Fig. 1a). A similar level of the drained treatment with PO93 was achieved only at day 42, while with P. ostreatus CBS 411.71 losses were lowest (139 g/kg at day 42). V. volvacea increased DM losses almost linearly from day 7 to 28 in the remoistened straw (y = 0.725x − 0.642, R 2 = 0.97, p = 0.002) to 192 g/kg DM at day 28 ( Fig. 1b). . The interaction of type of wetting and autoclaving or not was significant for ADFom, whose content increased both by soaking plus draining and by autoclaving ( Table 2). Parameters resulting from calculations including ADFom were equally affected. Most of the parameters were influenced by the type of wetting (Table 3). Draining led to higher EE, aNDFom and ADL contents while NDFD 30h and ELOS were reduced compared to remoistening. Autoclaving enhanced gas production slightly and increased ELOS (Table 4). When compared to the original straw, remoistening plus autoclaving increased the in vitro digestibility in terms of NDFD 30h , gas production and ELOS.
Biological treatment. There was a significant interaction of fungal strain and storage time of the drained straw for all three indicators of in vitro digestibility (Table 5). In the remoistened treatments, these indicators showed a uniform decrease starting in the second week and were less influenced by the fungal strain (Fig. 2). Although numerically higher, the NDFD 30h did not increase significantly during the first week after inoculation, both in the drained and the remoistened treatments ( Fig. 2a,b). Instead, it decreased in the course of time. Comparing drained and remoistened straw inoculated with P. ostreatus PO93 and C. subvermispora after 7 and 14 days, only NDFD 30h of C. subvermispora in the drained straw was significantly higher than its counterpart in the remoistened straw. ELOS and HFT values were comparable within the two fungal strains at the same time. At no time either of the in vitro digestibility parameters was improved compared to the starting point or the dry control straw (Fig. 2, grey baseline).

Discussion
Solid state fermentation of straw with basidiomycetes is an approach of valorizing field crop residues either for ruminant nutrition 10 or for human mushroom consumption 23 or biofuel production. Moisture, temperature, indigenous microflora are some factors, which influence both the growth of the fungi 24 , but also the nutritional composition and digestibility of the straw post-harvest 25 . To make uptake on farm likely, all processing steps have to be considered and minimized if possible. The same applies to losses from the field to the feeding trough.
Most of the studies on straw treatment with fungi soak the straw for several hours or days in abundant water and drain it then for several hours 11,26,27 . The presented study started with this following the recommendations for hobby mushroom cultivation 28 . However, in the chemical analysis it was realized that most of the fermentable carbohydrates got lost and that the lignin concentration increased by around 23% 17 with decreasing digestibility at the same time. That is why the pre-treatment was changed to watering the straw with a limited volume of water to get a dry matter of approximately 25%. According to Streeter et al. 29 a higher DM content (50%) was recommendable for incubation, which is in contrast to the observations by Abdullah et al. 30 who recommended 80% moisture content for optimal fungal growth.
A second point in the pre-treatment is autoclaving, which is a common practice in laboratory studies (e.g. 31,32 ). To make practical uptake on-farm more likely, this approach was abandoned.
To evaluate the effect of the physical pre-treatment a separate trial was performed. Remoistening plus autoclaving had the clearest effect on digestibility increase. Most advantages were seen in NDFD 30h (+ 17%) and ELOS (+ 12%). High-pressure steam treatment resulted in higher digestibility of DM and cell wall constituents with different roughages 33 , and even hot water at pH 4-7 had an effect by removing lignin and hemicellulose 34 .
The relation of cellulose/ADL was highest in the remoistened straw without autoclaving. However, this parameter was obviously less related to digestibility in contrast to the findings of Nayan et al. 11 , who determined a correlation of r = 0.64 between (cellulose + hemicellulose)/ADL and IVGP (in vitro gas production). The ratio of ADL/ADFom showed also no clear relationship to digestibility. The calculated forage quality parameters (TDN, RFQ, DMI) were similar between the remoistened and the original straw while they were lower for the drained treatments. This is linked to the formulae as they include both NFC contents and NDFD 30h . As NFC contents were significantly reduced by almost half due to leaching this had a major influence on the parameters. However, ELOS remained on the same level as the original straw.
In contrast to these findings, no digestibility increase was found in the fermented straw although NDFD 30h was numerically higher within the first 7 days on average. That was a reason for shortening the fermentation period to 14 days in Trial 3 and having a look at closer sampling intervals. www.nature.com/scientificreports/ In the presented trials, the same strain of C. subvermispora (CBS 347.63) was used as in other experiments performed in Wageningen. In those studies, the IVGP increased during a 7-week period of SSF based on drained and autoclaved wheat straw by around 30% although it was not compared to the original dry straw 11,27 . In any case, no increase in IVGP (HFT) was observed under the non-sterile conditions of the presented study. Decrease in digestibility in these trials went along with increase in lignin concentration by up to 42 g ADL/kg DM 17 . Nayan et al. 35 suspected a problem in the ADL analyses when handling mushrooms as they observed increased IVGP by 28-48% despite high ADL values. However, gas production did not increase in the presented trials. www.nature.com/scientificreports/ The P. ostreatus strain CBS 411.71 was earlier employed in an experiment for bioethanol production from wheat straw 36 . There, after 14 and 28 days, it improved enzymatic digestibility, increasing (hemi)cellulose digestibility from 35 to 55%. However, fermentable sugar yields were comparably low.
The generally observed decreasing digestibility in the presented trials might also be related to the lacking autoclaving in terms of sterilization prior to inoculation as the competing epiphytic microflora might have consumed the nutrients released by the white rot fungi similar to the observation made by Lang et al. 37 . Or fungal degradation was inhibited by an effect of competition with the native microflora 38,39 . In any case, Streeter et al. 29 stated that autoclaving in his small samples sizes was not necessary. Tuyen et al. 40 compared the gas production of straw inoculated with different fungi for up to 7 weeks to only autoclaved wheat straw (control). On day 21, out of 6 fungal species, C. subvermispora and L. edodes showed a higher gas production, on day 35, P. eryngii went beyond the control. However, the other species did not surpass the control line. V. volvacea inoculated straw declined linearly in IVGP from day 21 to day 49 of incubation 40 , which was more comparable to our observation.
For upscaling, Zadrazil et al. 7 worked with non-sterile culture conditions. However, the author did not present digestibility differences compared to sterile conditions. Also Rai et al. 41 seem to have worked without autoclaving using Coprinus fimetarius in rice straw and obtained results in feeding trials with goats in India, which were comparable to urea treated straw.
The temperature optimum for growth and metabolism differs from species to species and strain to strain. The temperature range for incubation from 21 to 24 °C applied in the presented study is similar to the one reported by Nayan et al. 27 , van Kuijk et al. 12 and Fazaeli et al. 42 , although it is lower for V. volvacea compared to Belewu and Belewu 15 with 35 °C for example. In any case, growth was observed for all inoculated strains, both under acidic and alkaline conditions 17 .
The treatment of draining and soaking alone led to about 10% DM losses and even higher losses in NFC concentration. Another 10% DM were lost within 14 days of incubation independent from the pre-treatment. Some studies quantify the different losses. With fungi of the genus Ionotus about 24% DM losses were observed after 30 days of incubation in wheat straw 43 . Zuo et al. 44 found up to 50% DM loss with Pleurotos chrysosporium in maize stover after 28 days, which was relatively high compared to other findings 14,45 . Although a certain increase in in vitro DM digestibility was achieved when incubating wheat straw with P. ostreatus and Erwinia carotovora, DM losses of 52% question the loss of 69% lignin which led to a slightly higher IVDMD in another study 46 . Besides, DM loss did not necessarily go along with the desired loss of lignin 11,43 and/or increase of digestibility as shown in the physical treatments here.
Some authors recommended a short fermentation period of 6-15 days to minimize losses 16,41,47,48 and to increase nutrient intake in vivo 41 . Besides, some white rot fungi have a high initial selectivity for lignin 49 . Shrivastava et al. 50 even found the highest in vitro OM digestibility after 5 days of SSF. That is one reason why in Trial 3 the fermentation time and the sampling intervals were reduced. However, it is in conflict with the different potential delignification phases described by Zadrazil et al. 7,25 and van Kuijk et al. 9 .

Conclusions
The study emphasizes the complexity of solid state fermentation with the purpose of ruminant nutrition. None of the tested fungal strains was able to improve the in vitro ruminal straw degradability under the given conditions. Pre-treatment gains the more importance the more it comes to upscaling. Moistening enables the fibrous complex to swell and thus be more easily attacked by the ruminal microbiome. As autoclaving is rather unrealistic for on-farm application, simple physical treatments such as the use of hot process water should be focused as a potential economical option to enhance ruminal fermentability of lignocellulosic materials.

Data availability
The datasets generated during the presented study are available from the corresponding author on reasonable request.